KEYWORDS
Hybridoma, genome-scale metabolic model, antiapoptotic gene,
mitochondrial transport, BHRF1
1.- INTRODUCTION
The demand for monoclonal antibodies has severally increased over the
last years, mainly for the new applications in therapy, but also for
clinical diagnosis and highly specific purification processes (Walsh,
2018; Grilo and Mantalaris, 2019). In this regard, the capacity of
mammalian cells to perform complex post-translational modifications to
yield biologically active proteins has led them to be the preferred
system for biopharmaceuticals production. About 70-80% of all
biopharmaceuticals, including monoclonal antibodies, viral vaccines and
gene therapy vectors, are produced with mammalian cells; and among the
top ten selling protein biopharmaceuticals in 2014, six of them are
antibodies or antibody-derived proteins. It is therefore not surprising
that monoclonal antibodies-based drugs production using mammalian
cell-based systems in the 2016 reached almost the double of the 2010
value (Estes and Melville, 2013; Walsh, 2018). In the last years,
monoclonal antibodies industrial manufacturing has been based on
mammalian cell lines such as hybridoma among others (Wurm, 2004;
Coco-Martin and Harmsen, 2008; Estes and Melville, 2013; Hnasko and
Stanker, 2015).
Mammalian cell-based processes present an important culture limitation:
the accumulation of metabolic by-products (i.e. lactate and ammonia) up
to cytotoxic concentration, as well as the depletion of essential
nutrients, triggers the apoptosis (programmed cell death) (Arden and
Betenbaugh, 2004). The prevention of apoptosis during the cell growth
has a critical effect on final process productivity: The increase of
cell life-span results in an increase of product of interest synthesis
and accumulation, since cells remain productive for a longer time, even
after the exponential cell growth phase (Zhang et al., 2018). Moreover,
more robust cell lines less sensible to apoptosis allow designing high
cell density culture strategies based on keeping low nutrients
concentration in narrower ranges (Casablancas et al., 2013).
In the last years, the strategies to generate stress-resistant cell
lines preventing apoptosis have been focused on blocking the apoptotic
transduction pathways (Smolke, 2010; Henry et al., 2020). Although there
are different pathways controlling the activation of signaling cascades
of cell apoptosis activation, many of the apoptosis signals converge on
the mitochondria, which stores numerous molecules that activate
apoptosis (Arden and Betenbaugh, 2004). The most used strategy to
prevent apoptosis has been the overexpression of bcl-2 or bcl-xL genes,
which inhibits the release of pro-apoptotic molecules from the
mitochondria (Smolke, 2010; Vives et al., 2003b). This strategy has been
successfully applied in different mammalian cell lines, as CHO or
hybridoma, showing higher viabilities and improved robustness in cell
culture (Fussenegger et al., 2000; Mastrangelo et al., 2000; Simpson et
al., 1997; Tey et al., 2000). The expression of mcl-1, another
antiapoptotic gene with similar mechanism to bcl-2/bcl-xL, has also
shown good results (Majors et al., 2009). Another approach has been
targeting directly the caspase cascade, expressing X-linked inhibitor of
apoptosis (XIAP) or cytokine response modifier CrmA, which both act
inhibiting directly the caspases (Sauerwald et al., 2003). The
expression of different viral proteins has also been reported to have
antiapoptotic effects in hybridoma cell cultures, as ksblc-2 from
Karposi’s sarcoma-associated herpesvirus (Vives et al., 2003a) and
bhrf-1 from Epstein-Barr virus (Juanola et al., 2009). Furthermore, the
downregulation of the pro-apoptotic genes Bak, Bax and Casp3 has shown
to reduce the apoptosis in CHO cells (Xiong et al., 2019).
Additionally, anti-apoptotic genes have shown an effect on metabolism,
although this is not fully understood yet (Dorai et al., 2009; Templeton
et al., 2014). This is remarkable as cell-based processes present an
important limitation regarding the metabolism: deregulated substrates
uptake (high consumption rates of mainly glucose and glutamine), what is
linked to the secretion and accumulation of lactate and ammonia as
by-products of the metabolism (Martínez-Monge et al., 2018a).
The reduction of the secretion and accumulation of lactate remains a hot
topic for biomanufacturing industry. Many different approaches have been
explored to reduce or delay lactate generation in cell culture including
media design by substitution of glucose for alternative carbon sources
like fructose or galactose (Altamirano et al., 2006), different
fed-batch strategies limiting glucose concentration (Casablancas et al.,
2013; Zhang et al., 2004) and several cell engineering approaches as the
expression of pyruvate carboxylase (Henry and Durocher, 2011) and
downregulation of lactate dehydrogenase (Chen et al., 2001). In all the
different scenarios described, a reduction of lactate accumulation up to
a certain extend was observed but never totally depleted.
In the present work, a murine hybridoma cell line (KB-26.5) has been
engineered to over-express BHRF-1 protein (KB26.5-BHRF1). Besides the
protective effect against apoptosis achieved, the over-expression of
BHRF-1 had an unexpected direct effect on cell physiology and
metabolism. Mainly higher cell growth rate and more efficient nutrient
usage were observed in batch cultures, significantly reducing the
lactate generation. Therefore, metabolic flux balances techniques were
applied to better understand the interactions and effects of BHRF-1
protein expression with cell metabolism. The interpretation of the
intracellular fluxes obtained allowed an understanding of their effects
on the improved cell metabolism and also allowed to arise some
hypothesis about the possible BHRF-1 interactions with the metabolic
pathways. Nowadays, prevention of apoptosis remains as a relevant
research topic as it has been addressed in a recent review by Henry et
al., (2020), in which the effect of different anti-apoptotic strategies
applied in the last years were presented and discussed.